Biochemistry: STRUCTURE

- Dr. Priyanka Halder Mallick, Associate Professor in Zoology, Vidyasagar University. Chemical Bonds holding Net protein structure = units + Chemical bonds Protein Synthesis a multiple dehydration process Types of Chemical bonds A. Primary bond:  Covalent : (Fischer, 1906)  It is the backbone of protein chain.  It is a specialized or an N-substituted amide linkage (– CO-NH-) B. Secondary bonds:  Many protein properties don’t coincide with the linear chain str. variety of bonds other than peptide exist in them.  They hold protein chain in its natural configuration. 1. Disulphide bond 2. 3. Non- polar / Hydrophobic bond 4. Ionic/Electrostatic bond Types of Chemical bonds 1. Disulphide bond:  Covalent bond formed by oxidation of thiol / sulfhydryl (-SH) gps. Of 2 cysteine residues yield a mole of Cystine, a.a. with a disulfide bridge.  Bond strength ~ 50 Kcal/Mole; bond length~ 2 Å between 2 S- atoms  To bring S-gps closer, -S-S- bond requires polypt chain to be folded.  -S-S- bridges are very strong w.r.t. non-covalent bonds but are very short ranged so that slight extension breaks them fully.   they stabilize 3o str after it has reached its final form.  E.g. Oxytocin, Insulin  General reaction:

2R-SH + ½ O2 R-S-S-R + H2O  Formation of Cystine :

HS - CH2 – CH – COOH Oxidation S - CH2 – CH – COOH O + NH2 NH2 HS - CH2 – CH – COOH Reduction S - CH2 – CH – COOH NH2 NH2 Cysteine (2 moles) Cystine (1 mole) Types of Chemical bonds 2. Hydrogen bond:  An energetically favorable interaction occurring when a gp. with a H i.e. covalently bonded to an electronegative e.g. O or N is in vicinity (2.8 Å) of a 2nd gp. containing an electronegative atom, is called a H-bond.  Tendency of H-atom to share e-s with 2 neighboring atoms esp. O & N leads to formation of a H-bond  E.g. carbonyl O-atom of 1 peptide bond shares its e-s with H-atom of another peptide. >C::O::H:N< or >C = O - -2.79 Å- - HN< (secondary valence bond)  Bond strength = 5 – 8 Kcal/mole maximal when linear bond  Binding energy = 1/10th of that of primary valence  H-bonds are relatively weak linkages but collectively exert considerable force that help in maintaining 2o str of proteins. Types of Chemical bonds Hydrogen bond:  E.g. Silk fibroin, Keratin of wool.

 Fig: common egs. of H- bonding:

a) 2 H2O mols. b) H2O & an

c) 2 amide gps. Types of Chemical bonds 3. Non- polar / Hydrophobic bond:  Hydrophobic side-chains or R gps of a.as unite among themselves to form linkages betn diff chains or betn various chain segments to eliminate water molecules like ‘coalescence of oil droplets suspended in water’ to form this bond.  Relatively strong bonding  Brings together gps that can form H-bonds /ionic bonds in absence of water, most efficiently & each type linkage helps in formation of the other.  Imp. role in other protein interactions: -  Enzyme – substrate complexes  Antibody – antigen interactions  E.g. it’s found in Ala, val, leu, met, trp, phe ala, tyr etc.  Fig. on page 8. Types of Chemical bonds

4. Ionic/Electrostatic bond / Salt – linkage/bridge: • When 2 oppositely charged gps are brought close together, electrostatic interactions lead to this strong attraction. • Long polypt chain contains large no. of charged side chains, giving many opportunities for such interactions. • Ionized gps. frequently stabilize interactions betn protein & other molecules ( than intramolecular ionic bonds) • Bond strength = weaker than H-bonds • Responsible for maintaining folded str. i.e. 3o str of globular proteins. • E.g. i) Divalent cations (Mg) – having 2 acidic side chains ii) Acid gps – basic gps of constituent a.as Positively charged gps Negatively charged gps

side chains of : - COO- gp of side chain of : -

• Lysine • Aspartic acid • Arginine • Glutamic acid • Histidine Types of Chemical bonds The Protein Structure INTRODUCTION

Proteins and peptides are biopolymers composed of amino acid residues interlinked by amide bonds. Their structure can be discussed in terms of four levels/degree of complexity of the molecule, as follows:

I. Primary Structure II. Secondary Structure III.Tertiary Structure IV.Quaternary Structure Basic structural levels of Organisation

• Subunits • Simpler • • Complete chain interact in a specific • Unfolded formation • Specific patterns • Similar / manner • Linear chains produce twisted 3-D Dissimilar • E.g. Hb molecule polypeptide oligomer subunits join to form larger proteins ABOUT PROTEIN FOLDING

1. Around 4000 structures known from X-ray crystallography and 2-D NMR studies

2. Structure database widely available for analysis

3. Water soluble proteins are "globular," tight packed, water excluded from interior, folded up.

4. Bond lengths and bond angles don't vary much from equilibrium positions.

5. Folding possibilities are limited, both along the backbone chain and within the side chain groups. ABOUT PROTEIN FOLDING

6. Structures are stable and relatively rigid.

7. Folding motifs are used repetitively.

8. Proteins with similar function typically have similar structure.

9. With similar proteins (say from different organisms) structure tends to be more conserved than the exact sequence of amino acids.

10. Although sequence must determine structure, it is not yet possible to predict the entire structure from sequence accurately. Basics…..

 POLYPEPTIDE - A long peptide chain (typically with mol. wt.<10,000).

 PEPTIDE - Two or more amino acids covalently linked by an amide bond between the carboxylic acid group of one and the alpha- amino group of the other.

 PEPTIDE BOND - The amide, covalent linkage of peptides.

 AMINO ACID - Polymers from alpha-amino carboxylic acids form proteins. Primary Structure

• Depends on the no. & str. of amino acid • Mode of linkage is - PEPTIDE BOND • Formulated by Linus Pauling & Robert Corey (1930s) • Rigid & planar bond Amide = 1.32 Å • C—N bond length Simple Anine = 1.49 Å • Associated atoms – Coplanar • Resonance – Partial sharing of 2 pairs of e--s betn carbonyl O & amide N small electric dipole is set. (fig. below) Primary Structure:

PEPTIDE BOND : • C, H, O, N of a peptide group lie in a single plane • Carbonyl O & amide N are trans to each other. • Virtually all peptide bonds occur in trans configuration. • 3 Covalent bonds between C – C – N - C •Amide C-N bonds are unable to rotate freely due to their partial double bond character making them rigid. • They comprise 1/3rd of the backbone bonds Primary Structure PEPTIDE BOND: Primary Structure PEPTIDE BOND:

• Fig. Details of the planar peptide bond Primary Structure AMINO ACIDS 18 of the 20 commonly occurring, genetically encoded amino acids involved in protein structure have a primary alpha-amino group and an optically active alpha-carbon centre. All also have an alpha hydrogen atom. The names of the amino acids then specify the fourth substituent attached at this position. AMINO ACIDS Formation of the peptide bond:

Thus the polymer formed is an unbranched chain of amino acids. Primary Structure CROSSLINKING IN PROTEINS The most common covalent crosslink between peptide chains is the disulfide bond formed by two cysteine residues.

In some connective tissue, protein crosslinks are formed by chemical modification. In collagen, lysine and histidine residues are used. In elastin the aromatic crosslinks, desmosine and isodesmosine, are formed from modified lysyl residues. Secondary Structure Definition of the torsion angles- phi, psi and omega  Folding occurs by Rotation about Single Bonds: Since bond length and angles are fairly invariant in the known protein structures, the key to protein folding lies in the torsion angles of the backbone.  A torsion angle is defined by 4 atoms- A, B, C and D. Secondary Structure Definition of torsion angles - phi ϕ, psi ψ, omegaω  When atoms A, B, C and D are main-chain atoms

(i.e. the carboxylic carbon, C1; the alpha carbon, C2 or C ; the amide group nitrogen, N; and another one ), there are THREE repeating torsion angles along the backbone chain called ϕ, ψ and ω. Secondary Structure Rotation about the amide bond  The OMEGA angle tends to be planar (0 or 180o) due to delocalization of the carbonyl pi electrons & the nitrogen lone pair. Trans is generally favoured over cis:

 Only 116 (0.36%) of 32,539 angles in 154 X-ray structures were found to be cis (Stewart et al. 1990).  However, some specific bonds are often cis, e.g.. Tyr-Pro (25%), Ser-Pro (11%), X-Pro (6.5%) This leaves phi and psi for flexible folding of the chain. However, steric conflicts limit even these angles as well. Secondary Structure Ramachandran plots and regular structure  By assuming that atoms behave as hard spheres, allowed ranges of ϕ, ψ can be predicted & visualized in steric contour diagram.  This plot of ϕ vs. ψ is called a Ramachandran plot (after G. N. Ramachandran) or conformational map. Repeating values of phi and psi along the chain result in regular structure. Secondary Structure Ramachandran plots and regular structure  Similarly, repetitive values in the region of ϕ = -110 to -140 and ψ = +110 to +135 give extended chains with conformations that allow interactions between closely folded parallel segments ( structures). The structure of plastocyanin is composed mostly of beta sheets and the Ramachandran plot shows a broad range of values in the - 110,+130 region. Secondary Structure

 It’s the local structure/ folding typically recognized by specific backbone Torsion Angles and specific main-chain Hydrogen Bond pairings that are of regularly spaced intrachain type.  It constitutes of helices that refer to steric or spatial relationship of a.as near to each other in the a. a. sequence.

◦ A helix is a rigid & tubular structure resulting from folding & H-bonding betn neighboring a.a. It involves regular coiling of molecules in the globular protein.

 Pauling & Corey (1951) observed periodicity in X- ray Diffraction studies implying regularity in protein structure. Secondary Structure Types of Secondary structures: based on nature of H-bonding in globular proteins

Regular or Repetitive Irregular

α- β- β-/ pleate Triple bend/ Random γ- helix d helix hairpin coil sheet bend/loo helix p Intra Inter Repulsion e.g. Insuffi- molecula molecula Reverse between ciently r H- r H- Collage turn of adjacent stable bonding bonding n polypeptide bulky residues Secondary Structure Regular structure: α - helix

 The is a polypeptide chain with planar peptide bonds forming a right-handed helical structure by simple twists about the α-C—N & α-C—COOH bonds (the α-C atoms have mobility)  α-helix is a rod-like structure, whose inner part is formed by tightly coiled polypeptide main chain & the side chains extend outward in a helical array.  Intramolecular/intrachain H-bonding stabilizes the α - helix spontaneously; H-bonds along the chain are almost co-axial  the main chain carbonyl C=O of ‘n’th residue is H- bonded to the amide N-H of ‘n+4’th residue along the chain Secondary Structure Regular structure: α - helix

 The α-conformation has well-defined dimensions .

 In the nominal range, phi (ϕ) = -60o & psi (ψ) = - 45o to -50o ◦ there are about 3.6 residues per turn of the helix ◦ Pitch=5.4 Å ◦ Identity period of helix = rise per residue =5.4/3.6 = 1.5 Å

 E.g., Myoglobin, Haemoglobin, Chymotrypsin, Cytochrome C, transmembrane proteins, α-keratin, etc. Fig: next page. Secondary Structure Regular structure: α - helix Fig: Average Dimensions of an a-helix Secondary Structure Regular structure: α - helix  3 D aspects of α - helix Secondary Structure Regular structure: α - helix  There are 2 types of α-helix -- left-handed & right-handed. Secondary Structure Regular structure: β pleated sheet  The β sheet is another min energy/ stable conformation characterized by interchain/ intermolecular H-bonds that help stabilize the str.  Formed by parallel alignment of a no. of polypeptide chains in a plane with H-bonds betn C=O & N-H gps of adjacent chains.  R-gps of constituent aa.s in a polypeptide chain alternatively project above & below the plane of sheet forming a 2-residue repeat unit.  β sheet strands are not fully extended (ϕ, ψ = - 180,180) due to sidechain steric interferences - the chain is slightly "puckered" so that the sheet is said to be "pleated" . Secondary Structure Regular structure: Beta pleated sheet Types of β sheet: Parallel Antiparallel Chains run from n-terminal to c- Direction of chains alternate terminal in the same direction On average, ϕ, ψ = -119,113 ϕ, ψ = -139,135 Repeat period is shorter =6.5 Å Repeat period is slightly greater = 7 Å

 Both conformations are otherwise similar  Sometimes pleated sheets can also be formed from a single polypeptide if the chain repeatedly folds upon itself.  E.g., structural proteins; enzymes - Lysozyme, Carboxypeptidase A; fibrillar proteins - Silk fibroin (antiparallel), etc. Secondary Structure Regular structure α-helix β-pleated sheet Conformation of Tightly coiled Fully extended polypeptide chain Axial distance betn 1.5 Å 3.5 Å adjacent a.a.s Stabilizing H-bonds Same strand of Different polypeptide betn NH & CO gps in- polypeptide strands

 Other structures with repetitive ϕ & ψ angles that are sterically allowed: Name phi psi Comments alpha-L 57 47 left-handed alpha helix 3-10 Helix -49 -26 right-handed pi helix -57 -80 right-handed Type II helices -79 150 left-handed helices formed by polyglycine and polyproline Collagen -51 153 right-handed coil formed of three left handed helices Secondary Structure Regular structure: Collagen

 Basic collagen monomer is a triple helix made of 3 αchains, each ~1000 residues long - left-handed helical str. - 3 residues per turn.  The aa sequence is remarkably regular - every 3rd residue is Gly & others are Ala, Pro, 4 HyPro (min+unique)  Rod shaped molecule, 3000 Å long, 15 Å diameter  Intrachain H-bonds absent but interchain H-bonds betn CO gp of another chain stabilize the 3 strands with the help of steric repulsion of pyrrolidone rings of Pro & Hyp residues.  3 strands wind around each other in a cable fashion to form a twisting in right handed pattern (opp of α keratin)  Such tight wrapping provides great tensile strength + no capacity to stretch. Secondary Structure Regular structure: Collagen Triple Helix

• Collagen fibrils consist of recurring 3- stranded polypt units - Tropocollagen - arranged head to tail in parallel bundles - may be homo- or hetero-trimer • Heads of adjacent tropocollagen molecules are staggered & allignment of the head gps of every 4th molecule produces char. cross-striations 640 Å apart • A series of complex covalent cross-links are formed within tropocollagen molecules in the fibril, leading to formation of strong mature collagen. Secondary Structure Regular structure: Collagen Triple Helix Secondary Structure Regular structure: Turns  Several definable turns and bends in protein structure have been recognized and classified either by the relationship between the ϕ, ψ angles of the residues in the turn or the hydrogen bonding of their amide N-H and carbonyl-oxygen atoms:- 1. β turn or β bend or Hairpin bend: ◦ Polypeptide chain abruptly reverses dirn, leading to compact globular shapes ◦ A tight turn about 180o involving 4 aa residues ◦ Bend formation due to H-bonding betn CO gps of residue 1 & NH gp of residue 4. & proline are commonly found. ◦ They usually connect ends of 2 adjacent segments of an antiparallel β-sheet ◦ β turns are oft found near surface of a protein & also called Reverse turns. ( Fig: next page) 2. Gamma (γ) turns: ◦ The tightest turns involve only 3 residues with hydrogen bonding between the carbonyl of the 1st residue (N-terminal end) and the N-H of the 3rd residue. ◦ The centre residue has ϕ, ψ values near 80,-65 (or -80, 65 for inverse gamma γ turns), a region of the Ramachandran plot not typically occupied. Secondary Structure Regular structure: Turns Secondary Structure Irregular structure

1. Random coil  Polypt containing adjacent bulky residues such as Isoleu or charged residues like glutamic a. & aspartic a. undergo repulsion betn these gps- leading to random coil configuration  Thus, R-gps distributed along polypt backbone determine ◦ 2o str adopted by diff portions of it. ◦ Lack of a well defined str (fig.)

2. γ-helix  Highly H-bonded str  Lack interatomic contacts => insufficiently stable to be used in proteins. Tertiary Structure Characteristics  Tertiary Structure is the folding of the total chain, the combination of the elements of secondary structure linked by turns and loops. Its stability is determined by non-bonding interactions & the disulfide bond.  The tertiary structure of proteins is characterized by tightly folded structure with polar groups on the surface and non-polar groups buried. Natural variation from species-to-species tends to favour changes in surface (and therefore polar) groups.  Globular proteins exist - additional bonds at regular intervals in the helix - otherwise elongated strs + small cross section  Globular proteins like enz, transport protein, some peptide hormones, Ig possess hydrophobicity created by interactions betn polar gps located on molecule’s exterior+ non-polar R-gps in interior  Thus, folding of helices of globular proteins Refers to - the spatial arrangement of a.a.s far apart in linear sequence & pattern of disulphide bonds Tertiary Structure E.g., Myoglobin Tertiary Structure Folding motifs of Proteins  There are a number of ways to represent the folding of a protein and the arrangement of secondary structure elements within the tertiary structure.  Examination of such diagrams reveals recurring structural patterns in protein folding, while these don't show the sidechain and mainchain interactions that hold the structures together.

o  Fig: Simplified 3 folding pattern Red = helices, Green= β sheets Tertiary Structure Folding motifs At the simplest level, proteins can be classified by their content of secondary structure. Typically, the focus is on α-helices and β-sheets. However, there are some proteins where turns and disulfide bonds seem to be more important considerations. E.g. Wheat Germ Agglutinin, where 16 turns (in cyan) and 16 disulfide bonds (in brown) are the predominant structural contributions. While a large part of the structure has no classified secondary structure (in black), there are still four structurally homologous domains related by symmetry. Tertiary Structure Folding motifs

 Some proteins are made  Some are mostly β- up of mostly α-helices. sheet. E.g.

marine E. coli cyto- The green alga sea snake bloodworm chrome B562 plastocyanin neurotoxin haemoglobi (4 helix bundle n common motif) Tertiary Structure Folding motifs

 Many proteins are a mix of 2o components: α- helices and β-sheets.

Pancreatic Trypsin β-sheet core of H-RAS P-21 Ribonuclease T1 Inhibitor Carbonic protein (beta Anhydrase saddle)

β-barrel found in the Triose Phosphate Isomerase (left) and Xylose Isomerase (right) structures Quaternary Structure Characters

 Quaternary Structure is the combination of two or more chains, to form a complete unit. The interactions between the chains are not different from those in tertiary structure, but are distinquished only by being interchain rather than intrachain.  4th degree of complexity - great value in many proteins  Some globular proteins consist of 2 or more interacting peptide chains, each called a subunit.  These chains may be identical or diff. in their primary str. o  The same forces involved in formation of 3 strs. are also involved here.  The specific association of a no. of subunits into a complex large-sized molecules is referred to as 4o str i.e. it refers to the spatial arrangement of subunits & the nature of their contact. Quaternary Structure E.g. Haemoglobin molecule Quaternary Structure